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Home , Growth hormone, Secretin, Somatostatin

(M3 1 -3998/9413603-0315$03.o()/O PEDIATRIC RESEARCH Vol. 36. No. 3, 1994 Copyright O 1994 International Pediatric Research Foundation. Inc. Printed in U.S.A.

Somatotropic Dysfunction in Growth -Releasing Hormone-Deprived Neonatal Rats: Effect of Replacement ~hera~~'

SILVANO G. CELLA, VITO DE GENNARO COLONNA, VITTORIO LOCATELLI, GILBERT0 E. BESTETTI, GIAN LUIGI ROSSI, ANTONIO TORSELLO, WILLIAM B. WEHRENBERG, AND EUGENIO E. MULLER Department of Pharmacology, Chemotherapy and Toxicology, University of Milan, 20129 Milan, Italy [S.G. C., Kd. G.C., K L., A. T., E. E. M. I; Itutitlct fur Tierpathologie, Universitat Bern, CH-3001 Benz, Switzerland [G.E. B., G.L. R. I; Department of Health Sciences, University of Wisconsin, Milwaukee, Wisconsin 53201 [W. B. W.

In a previous work, we reported that passive immunization than in normal rat serum-GH-treated pups, whereas the total with anti-growth hormone-releasing hormone (GHRH) anti- GH immunoreact~typer unit surface, an index of intracellu- bodies (GHRH-Ab) in neonatal rats caused disruption of so- lar hormone concentration, was slightly higher than in vehi- matotropic function that was still present 60 d posttreatment. cle-GH or GHRH-Ab pups. As determined by electron mi- We studied the reversibility of this condition by growth hor- croscopy, somatotropes from GHRH-Ab+GH pups had mone (GH) replacement therapy. Neonatal rats received morphologic features of high cellular activity. It appears that in GHRH-Ab (50 ~Urdt,s.c.) or normal rabbit serum every GHRH-deprived pups GH replacement therapy can normalize second day from birth up to postnatal d 10 and received hGH most but not all altered indices of the somatotropic function. (0.4 &g body weight, s.c., bid.) or vehicle in a 2 x 2 factorial The effects of GH are mainly directed at the pituitaly, whereas design. Animals were studied on d 11 of age. In GHRH-Ab- the sensitivity of the to GH replacement is treated rats, GH therapy 1) counteracted the reduced body lower. (PediatrRes 36: 315-322, 1994) weight and low plasma IGF-I levels; 2) failed to modify the reduced pituitary weight and GH content; 3) further reduced Abbreviations the low plasma GH levels; 4) partially restored the defective GH, growth hormone GH responsiveness to GHRH; 5) failed to modify the reduced GHRH, growth hormone-releasing hormone hypothalamic and increased GHRH expres- GHRH-Ab, anti-GHRH-antibodies sion in the hypothalamus; and 6) reverted the decreased pitu- NRS, normal rabbit serum itary somatostatin binding. Morphologic and morphometric dCTP, deoxycytidine triphosphate evaluation of the from GHRH-Ab+GH pups PRL, showed that the number of GH-labeled structures was lower SS, somatostatin

Recent studies by us (1,2) and by other groups (3,4) have It is presently unknown whether these changes are due to shown that rat pups treated with GHRH-Ab may represent a the GHRH deficiency itself or to the consequent reduction of suitable animal model for the study of the human growth GH secretion. Therefore, we studied the effects of concom- disorders caused by a primary hypothalamic dysfunction. itant GH and GHRH-Ab treatment. We have administered Neonatal administration of GHRH-Ab permanently inhibits GH daily to GHRH-deprived and control pups from postna- the growth rate and alters several indices of the hypophyseal tal d 1 to 10 and subsequently evaluated in vivo and in vitro somatotropic function (I). several hypothalamic and pituitary indices of somatotropic activity. Received November 11, 1993: accepted March 21, 1994. Correspondence and reprint requests: Eugenio E. Muller, MD. Department of METHODS Pharmacology. Chemotherapy and Toxicology. University of Milan, via Vanvi- telli 32. 20129 Milan. Italy. All procedures were reviewed and approved by the Supported by a grant from Kahi Pharmacia, Italy. 'This study was executed under the auspices of the Itallan Society of Endocri- ethical committee of the of Pharmaco'og~of nology. GH study Group. the University of Milan. Functional Studies group were collected in pools of three samples (four pools per experimental group), immediately frozen on dry ice, Animals. Pregnant Sprague-Dawley rats (Charles River, and stored at -70°C until used. Total RNA was obtained Calco, Italy) were purchased and housed under controlled by single-step acid guanidium-phenol-chloroform extrac- conditions (22 k TC, 65% humidity, and artificial light tion (9). Total RNA samples (20 pglsample) were elec- from 0600 to 2000 h). After birth, all litters were culled to trophoresed on 1.2% formaldehyde-agarose gel and a standard size of 12 pups and left with dams during the transferred to a nitrocellulose membrane (Schleicher & experimental period (see below). Studies were conducted Schuell, Dassel, Germany) at room temperature for 24 h on d 11 of age. In all, 250 pups were used. in 10 x SSC (1 x SSC = 0.1 M sodium chloridel0.01 M Antiserum to GHRH. The GHRH-Ab was prepared by sodium citrate). Filters were hybridized with a rat GH immunizing rabbits with a mixture of synthetic rat GHRH cDNA sequence kindly provided by Dr. Frances DeNoto (5) and methylated BSA emulsified in Freund's adjuvant, (University of California, San Francisco, CA). The probe as previously described (6). The biologic efficacy of the was labeled using the Megaprime DNA labeling system antiserum was assessed at various levels. The GHRH-Ab (Amersham, Little Chalfont, UK) with [a-"PI~CTP to a has repeatedly been shown to significantly inhibit GH sp act of 1 x 109 dpmlkg DNA. Control of the homoge- secretion and growth (3, 4, 7). In addition, the antiserum neity of the amount of RNA loaded was performed by was tested for rat GHRH-binding capacity with reprobing the filters with [a-3'~]d~~~-labeledP-actin labeled rat GHRH. The antiserum dilution required to cDNA. After hybridization, autoradiography was carried bind 30% of the tracer was approximately 1.30 000. Char- out at -70°C for 24 h in the presence of an intensifying acterization of the antiserum showed that it was directed screen using Hyperfil-MP (Amersham). Quantification of toward the GHRH carboxy terminal. It cross-reacted the hybridization signal was performed on a scanning with synthetic human, bovine, and porcine GHRH by densitometer (LKB XL Laser Densitometer, LKB, Upp- less than 4%, and the dose-response curves were not sala, Sweden). Pituitary GH mRNA levels were ex- parallel with rat GHRH. pressed as a percentage of the control values. The antiserum did not cross-react with that Pituitary SS binding. The glands of have considerable with GHRH, in- seven pups for each experimental group were excised and cluding , , vasoactive intestinal , frozen in liquid nitrogen. The SS autoradiography was , , , and . performed on 14-km-thick, slide-mounted frozen sec- Treatments. Each single litter was divided randomly tions as described by McCarthy and Plunkett (10). Due to into two groups, one of which was given GHRH-Ab (50 the limited amount of tissue, only two concentrations of ~Llrat,s.c.) starting on d 1 and then on d 2, 4, 6, 8, and ["'I]T~~'-SS(2000 Cilmmol, Amersham) were used. One 10; the other group was given equal amounts of NRS. was 0.4 nM, near the k,, and the other was 0.8 Each group, in turn, was divided into two subgroups, nM, a saturating concentration for the receptors (11). which were given twice daily (at 0900 and 1900 h) hGH Nonspecific binding was determined with 10 pM unla- (Genotropin, Kabi Vitrum, Stockholm, Sweden, batch beled SS (Sigma Chemical Co., St. Louis, MO). Sections 870301, 0.4 kg/g body weight, LC.) diluted in normal were apposed to [3~]~yperfilm(Amersham), exposed for saline or equal amounts of normal saline from d 1 to 10. 3 d at -20"C, and quantified by using a computer-assisted All experiments were performed on d 11, at 0900 h, i.e. 14 image analyzer. The results were expressed in arbitrary h after the last GH injection. densitometry units. Eg'ct of acute GHRH. At 0830 to 0900 h, the free- Hypothalamic GHRH and SS . Hypotha- moving 11-d-old rats were injected with GHRH (human lami of the same pups used for pituitary GH mRNA GHRH-44, Sanofi, Paris, France, batch DADJOI) at the expression were collected. The ablated areas extended dose of 0.2 kg/kg body weight, S.C. and were killed 15 min from the optic chiasm to the mamillary bodies and later- later. Trunk blood was collected into EDTA-containing ally to the fornix. They were collected in pools of two tubes. Plasma samples and anterior pituitaries were samples, immediately frozen on dry ice, and stored at stored frozen until GH was determined by RIA, as pre- -70°C until used. The total RNA was isolated from viously described (8). In this study, about 50 pups were pooled hypothalamic tissue as alluded to previously (9). used. The total RNA samples (20 pglsample) were spotted onto IGF-I MA. Plasma IGF-I concentrations were evalu- nitrocellulose sheets (BA 85, 0.45 km, Schleicher & ated by a homologous RIA in plasma extracted with 2N Schuell; prewetted with 5 x SSC) using a slot-blot appa- HCI, 12.5%, plus 87.5% ethanol, using reagents provided ratus (Minifold 11, Schleicher & Schuell). Filters were by National Hormone and Pituitary Program (NHPP). hybridized with a rat GHRH cDNA (plasmid prGRF 2, The sensitivity of the assay was 100 pg/mL; intra- and kindly provided by Dr. Kelly E. Mayo, Northwestern interassay variabilities were less than 10%. The plasma University, Evanston, IL) and rat SS cDNA (plasmid levels of IGF-I of eight pups for each experimental group pSR-1, kindly provided by Dr. Richard H. Goodman, were determined. Oregon Health Center, Portland, OR). Specificity of hy- Pituitary GHgene expression. For the evaluation of GH bridization of these probes has already been tested (12, mRNA levels, 12 pituitaries from each experimental 13). Both cDNA probes were labeled with [a-"PI~CTP GH REPLACEMENT IN GHRH-DEPRIVED RATS 317 as previously described for GH gene expression. Control antisera are known to have negligible cross-reactivity for of the homogeneity of the amount of RNA loaded was other . The immunoreaction specificity was performed by reprobing the filters with [a-"PI~CTP la- evaluated by either omitting the primary antiserum or beled p-actin cDNA. Autoradiography was carried out at preadsorbing it with 10 wg/100 FL PBS purified rat GH or -70°C for 24-72 h with an intensifying screen (see PRL (UCB-Bioproducts, Brussels, Belgium) (17). above). Quantification of the hybridization signal was Furthermore, on one section per animal the antiprolif- performed as previously described, and the results were erating nuclear antigen (PCNA, BioGenex, San Ra- expressed as the percent of the control values. mon, CA) immunocytochemical reaction was performed Plasma and pituitary GH. Plasma and pituitary GH con- to detect mitotic processes. The reaction was revealed by tent were determined in six to 10 pups for each treatment a biotin-streptavidin-alkaline phosphatase supersensitive group by a double antibody RIA (14). Results are ex- detection system (BioGenex) (18). pressed as ng/mL and pg/pituitary in terms of the National Thin sections (60 nm) containing the entire hemipitu- Institutes of Health standard rat GH RP-2, the potency of itary profile were then cut from postfixed and nonpost- which was 2.0 IU/mg. The sensitivity of the assay was 0.5 fixed blocks for the electron microscopic studies. Sec- ng/mL; intraassay variability was 5%. To avoid possible tions from postfixed tissue were conventionally stained interassay variation, all samples of a given experiment for contrast by uranyl acetate and lead citrate. The im- were assayed in a single assay. munocytochemical GH labeling- was performed accord- ing to a previous protocol (16) modified as follows: no Morphologic, Morphometric, and Densitometric Studies etching, incubation in rabbit anti-human GH (Dako General. Groups and treatments were as described ~or~.)1:200for 1 h, incubation with goat anti-rabbit IgG above for the functional study. Six rats per group were conjugated to 15-nm colloidal gold particles (IG; Janssen used. Under intraperitoneal ketamine hydrochloride an- Life Science Products, Beerse, Belgium) diluted 1:40 in esthesia, whole-body perfusion-fixation was performed PBS-Tween-20 1:200, and washing in PBS-Tween-20 as previously described (15). 1:200. All thin sections were examined using a Zeiss Tissue processing. The was removed from the EM-902 PC (Carl Zeiss, Zurich, Switzerland) at 80 kV. skull and weighed. After extraction from the basal Densitometry. Densitometry was performed using a vid- meninges, the pituitary gland was weighed (Table 1) and eo-based, computer-linked system programmed to iden- cut longitudinally in half along the sagittal axis; a 0.5-mm- tify and measure OD in the selected images. Screen thick frontal slice was cut from each half at the level of displays of the amount of the immunoreactive substance the maximal pituitary height to yield two standard tissue in gray tones as well as the automatic measuring and blocks per animal, one for conventional, the other for evaluation of particles whose gray tone distinguished immunocytochemical studies, as previously described them clearly from the background were the main func- (16). Tissue blocks for conventional microscopy were tions of the program. Furthermore, the interactive editing postfixed with 2% phosphate buffered (pH 7.4) OsO,, of images allowed elimination of artifacts. those for immunocytochemistry were not postfixed. The immunoreactive material in the semithin sections After dehydration, infiltration, and embedding in was quantified as previously described (19), with modifi- Spurr's low viscosity medium, semithin sections (1 -5 pm) cations: the light setting was 200 lux and the images were were cut. Sections of each pituitary gland were stained taken by a 40x objective. In one standard frontal hemi- with toluidine blue for light microscopic study. The im- section, the immunohistochemically labeled cytoplasmic munocytochemical labeling of the hormones was per- portions of somatotrophs and lactotrophs were mea- formed by the peroxidase-antiperoxidase method, as pre- sured. viously described (15). We used rabbit anti-human GH The following measurements were obtained: 1)number and rabbit anti-human PRL (Dako Corp., Santa Barbara, of immunocytochemically labeled anatomical structures, CA), both diluted 1:200. These commercially available which roughly corresponds to the cell number; 2) total

Table 1. Body and pituitary weight and GH content in 11-d-old pups given NRS or GHRH-Ah and saline or GH replacement rherupv Pituitary weight GI1 content 111 vivo treatment* Body wt (g) mg mgi% body wt pgipituitary pgimg pituitary NRS + saline 19.1 + 0.3 2.1 ? 0.07 1 1.O + 0.4 18.8 ? 1.8 8.9 ? 0.8 NRS + GH 21.5 c 1.0t 2.1 -c 0.1 9.7 + 0.3 17.8 + 0.8 8.6 ? 0.6 GHRH-Ab + saline 17.6 2 0.5t 1.2 c 0.04$ 7.0 + 0.3$ 1 1.2 + 0.2$ 9.1 c 0.4 GHRH-Ab + GH 18.3 ? 0.4 1.3 ? 0.1% 7.2 + 0.6$ 11.2 2 1.5$ 9.2 -t 1.7 * NRS or GHRH-Ab (50 ~Llrat)was injected s.c. on d 1, 2, 4, 6, 8, and 10 of age. GH (0.4 pgig body wt) was injected s.c. twice daily from d 1 to 10. Each value is the mean + SEM of six to 10 determinations made in duplicate. The same description applies to suhsequent tables. See text for details. t p < 0.05 1,s NRS + saline value. $p < 0.01 vs NRS + saline value. 318 CELLA ET AL. area of the immunoreactive substance; and 3) number of In rats given GHRH-Ab, irrespective of concomitant pixels per gray level. Subsequently, we calculated the GH treatment, the total pituitary GH content, but not the product of the number of pixels times the corresponding GH concentration (~glmggland), was reduced (Table 1). gray level; this value, named "total immunoreactivity," Eflect of acute GHRH. Baseline plasma GH concentra- is considered a relative quantitative evaluation of the tions were significantly lower in GHRH-Ah-treated than antigen content (20). We also calculated immunoreactiv- in NRS-treated rats. GH treatment did not modify base- ity per unit area and per structure. Five windows were line plasma GH concentrations in NRS-treated pups but measured in each rat. further reduced plasma GH levels in GHRH-Ah-treated pups (Table 2). Statistical Analysis A single dose of GHRH did not raise plasma GH In the functional studies, mean values for experimental concentrations in GHRH-Ah-treated pups. Administra- and control groups (mean 2 SEM) were compared by t tion of hGH inhibited the GH response to the GHRH test after analysis of variance. A probability of p < 0.05 challenge in NRS-treated pups but partially restored it in (two-tailed) was taken to indicate a significant difference GHRH-Ab-treated pups (Table 2). among groups. Plasma IGF-I concentrations. Circulating IGF-I concen- In the morphologic studies, the significance of differ- trations were lower in GHRH-Ab-treated pups than in ences among groups was calculated by the Kruskall Wal- NRS-treated ones. Administration of GH increased sig- lis H-test. nificantly plasma concentrations of IGF-I in NRS-treated pups and restored them to normal in GHRH-Ab-treated RESULTS pups (Table 2). Growth rate. Until postnatal d 4, there were no differ- Pituitary GH gene expression. When total RNA was ences in growth rate among experimental groups. Be- subjected to Northern blot hybridization, GH mRNA was tween postnatal d 5 and ll, GHRH-Ab-treated rats had a detected as a single 800-bp band. growth rate significantly lower than NRS-treated rats. Pituitary GH mRNA levels were significantly (p < GH treatment increased body weight gain in NRS- 0.01) reduced in GHRH-Ab-treated pups (-54.6 2 2.0% treated pups and prevented the reduction in growth rate versus NRS-treated group). Administration of GH did not induced by GHRH-Ab. In NRS+GH-treated pups, the modify pituitary GH mRNA levels in GHRH-Ab-treated growth-promoting effect of GH became evident starting pups (-51.5 2 1.5% versus NRS-treated pups) (Fig. 2). from postnatal d 6, when their growth curve diverged Pituitary SS binding. SS receptor binding was signifi- significantly from that of pups given NRS alone. In cantly lower in the pituitary glands of GHRH-Ab-treated GHRH-Ab-treated pups, substitutive GH therapy com- pups than in those of NRS-treated animals. Administra- pletely counteracted the growth inhibitory effect of tion of GH did not modify pituitary SS binding in NRS- GHRH-Ab, so that no difference between their body treated pups but restored it to normal in GHRH-Ah- weight and that of NRS-treated pups was observed (Fig. treated pups (Table 3). 1). Hypothalamic GHRH and SS gene expression. Increased Pituitary weight and GH content. Both the absolute and hypothalamic GHRH mRNA levels and decreased SS relative (% body weight) weight of the pituitary gland mRNA levels were present in GHRH-Ab-treated pups were significantly reduced by GHRH-Ab treatment. Ad- (Table 4). Administration of GH did not modify GHRH ministration of GH did not modify these indices in NRS- and SS gene expression in either NRS- or GHRH-Ab- and GHRH-Ab-treated pups (Table 1). treated pups (Table 4). Morphometry, densitometry, electron microscopy, and im- munocytochemistry. The number of pituitary GH-labeled structures was lower in pups treated with GHRH- Ah+GH than in those given NRS+GH (Fig. 3A and C; -m NRS+GH - 20 Table 5). In contrast, the total immunoreactivity per unit F NRS + Sa(ine I GHRH-Ab+GH surface was increased, although not significantly, in 0 GHRH-Ab+ Saline GHRH-Ab+GH-treated compared with GH- or GHRH- $ l5 Ab-treated pups (Table 5). In the GHRH-Ab+GH- 10 treated group, the immunoreactivity was confined to the m cell periphery (Fig. 3C). The number of PRL-labeled structures was increased 5 1234567891011 in GH- and GHRH-Ab+GH-treated pups, whereas the AGE (days) PRL immunoreactive area and the total immunoreactiv- ity were increased only in the GHRH-Ab+GH group Figure 1. Changes in body weight with age in pups given NRS or GHRH-Ab and saline or GH replacement therapy. From the asterisks (Table 6). and following the arrows, values are significantly different @ < 0.01) No cell was labeled by the antiproliferating cell nuclear from those of NRS+saline-treated pups. antigen immunocytochemical reaction. GH REPLACEMENT IN GHRH-DEPRIVED RATS

Table 2. Basal and GHRH-stim~tlatedGH levels and circulating IGF-I concentratiorls in 11-d-old p11p.s gi\>en NRS or GHRH-Ab and saline or GH replacement therapy GH (ngimL)'

It1 titvo treatment Basal GHRH IGF-I* (ngimL) NRS + saline NRS + Gt1 GHRH-Ah + saline GHRH-Ah + GI4 * Hormone concentrations determined 24 h after the last GI1 treatment. t p < 0.01 trs respective basal value. $ p < 0.05 1:s NRS + saline value and p < 0.01 tr.s GHRH-Ah + saline value. $p < 0.01 1,s NRS + saline value.

By electron microscopy, somatotroph cells from pups has also been evidenced more recently (25-27). In the treated with GHRH-Ab+GH had small and marginated present study, the ability of GH replacement therapy to granules and abundant rough endoplasmic reticulum reverse some defective indices of somatotropic function (Fig. 3B and D). This finding was obtained on the con- in GHRH-deprived 10-d-old rats reinforces the view that ventionally postfixed preparations and confirmed by the somatic growth is pituitary dependent very early in the study of comparable cells on GH-labeled thin sections. postnatal period. The most overt effect of GH replacement therapy was DISCUSSION its ability to increase body weight gain in NRS-treated rats and restore to normal the defective growth rate of Earlier reports suggested that GH cannot affect growth GHRH-Ab-treated rats. The dose used was selected in the immediate postnatal period of several mammals based on its capacity to restore weight gain in hypophy- (21) despite its very high concentrations in the circulation sectomized rats (28). at this age period (22-24). However, in a previous study Although administration of GH also increased signifi- (1) we showed that in the rat passive immunization cantly plasma levels of IGF-I both in control and in against GHRH during the early postnatal period has GHRH-Ab-treated rats, the current data do not allow us profound and long-term effects on subsequent somatic to address the question of whether the growth-promoting growth and somatotropic function. Partial pituitary de- effects of GH were mediated by the increased plasma pendence of growth, even at the earliest neonatal stage, IGF-I levels. It has been shown that, in the rat, a poor

Table 3. A~ttoradiographicevahtution of ['"I]Tyr-1-SS binditzg to pituitary glands from 11-d-old pups given NRS or GHRH-Ah and salirle or GH replacement therapy ['2CI]Tyr-I-SSconcentration'

It1 ~~it~otreatment 0.4 nM 0.8 nM

NRS + saline 134.2 rt 9.1 361.2 s 15.3 NRS + GH 177.3 ? 21.5 439.5 ? 51.9 GHRH-Ah + saline 23.0 s 10.8t 232.9 s 46.9$ GHRH-Ab + GH 76.1 2 11.45 514.1 -c 39.35 * Results are expressed as arbitrary densitometry units; each value is the mean ? SEM of tive determinations made in quadruplicate. tp < 0.01 rJs NRS + saline value. $p< 0.05 vs NRS + saline value. $p < 0.01 vs GHRH-Ah + saline value.

Table 4. GHRH and SS gene expression in 11-d-old p~tps given NRS or GHRH-Ab and saline or GH renlacement theranv NRS NRS GHRH-Ab GHRH-Ab + + + + GHRH SS Sal~ne GH Sal~ne GH In r8ir~otreatment (mRNA %)* (mRNA %)* NRS saline 100 100 Figure 2. GI1 mRNA levels in pups given NRS or GHRH-Ab and + NRS GH +6.O 5 3.6 -4.7 5 1.6 saline or GH replacement therapy. The upperpunel shows representa- + GHRH-Ab saline +69.2 s 3.9t -44.7 ? 2.2t tive Northern blot hybridization signals from each experimental group + GHRH-Ab GH +67.2 5 3.9t -41.1 5 2.4t (20 Kg total mRNNlane). The hybridization patterns for rat a-actin are + also shown. The lower purlel shows the mean rt SEM of all the * Each value is the mean ? SEM of tive determinations; data are observations (four per group). *, p < 0.01 r7s NRS+saline and expressed as arbitrary densitometry units. NRS+GH values. t p < 0.01 1s NRS + saline and NRS + GH values. .anleA auges + qv-~~~f)SA 10.0 > d$ 'anleh HE) + SXN st%SO'O > d I. -yelap iaqlinj JOJ spoqlam aas .y a[qcL 01 sa!ldde uo!ld!~ssap awes aqL .&!~!)~ea~ounmm!lelol '1~.NZJS s ()CJ ad s~opu!~aAy jo wns) sueaw ale sanleA , $41'0 + I'EOI U'PI + P'Y91 Z'EY + 9LE 6'65 + PYE 48'ZE + 822 HD + qV-HtIHf) O'l i1'86 8'81 + P'OOZ L'IS 7 ZZS L'ES 7 6ZS 1'0P + ELZ au!les + 9V-HtIHf) 9'0 + E'66 0'6E T S'XOI Y'OZI + SEE 5'611 7 SEE Z'EI 7 IEE HD + StIN E'l + 0'101 Z'ZY + 6'EYI O'S01 % OLE S'L6 T 0YU I'EP 7 E9Z au!les + StIN

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-1uepunqe aeu~als!smnln~!ja~ ~!wseldopua q%no~ aql pue paleu!%ielu ale salnue~8aql :sdnd paleai)-~t)+s~~JO asoqi ueql sa(nuei8rOola~~as lallews aAeq pue snoialunu ssal ale sdnd palea~l-~f)+qv-~~~f)moij slla3 aqL ' x0()0~ 'Ado~soi~!~uoilJala leuo!luahuoJ :a'g : x~f~ 'uo!lJeai aseleqdsoqd au!lqle HD-que 'kdosso~s!w lq8!1 :3 'v '(apue 3) dd" paieaii-~t)+qv-~~~pue (g pue HD+S~NJO (s)sadoiloiewos 'E a~n%!d GH REPLACEMENT IN GHRH-DEPRIVED RATS 321

Table 6. Morphometty and immunodensitometty of lactotropic cells from 11-d-old pups given NRS or GHRH-Ah and saline or GH replacement therapy No. of structures Area (B) TI (C) h rir90 treatment (A) (x 10') (pn' x 10-I) ( x 10-7 CIA

NRS + saline 60 ? 10.9 20.2 ? 5.2 22.0 ? 5.8 34.5 ? 4.4 104.4 ? 0.7 NRS + GH 119 + 19.9' 50.3 + 14.8 54.6 + 16.5 45.3 * 9.4 107.8 2 0.8 GHRH-Ah + saline 113 ? 23.1 42.6 ? 11.7 46.4 ? 12.8 37.6 ? 3.9 108.6 ? 0.5 GHRH-Ab + GH 180 * 33.3t 50.5 2 12.6' 53.9 + 13.67 28.7 2 1.8 106.4 2 0.6 * p < 0.05 tjs NRS + saline value. tp < 0.01 rs NRS + saline value. release (35) but also GH gene expression (36) and tran- GH treatment do not allow a definite conclusion. Measure- scription (37). Fetal and neonatal pituitary cells in vitro ment of pituitary GHRH receptors may provide meaning- are already sensitive to this pituitary-directed inhibitory ful information in this context. It is noteworthy, however, action of IGF-I (38). that evaluation of the density of SS pituitary receptors, A direct effect of GH on the pituitary to inhibit its own although confirming that GHRH-Ab treatment markedly secretion is not likely, at least in adult rats (39). Also, the reduced this index of somatotropic function (1) and show- possibility that GH per se (40, 41) or through circulating ing that GH per se did not affect it, disclosed, most or in situ IGF-I (42,43) may act at the hypothalamic level importantly, that GH treatment counteracted the inhibi- is unlikely (see below). tory effect of GHRH-Ab. We do not know whether the We have previously shown (1) and confirmed in this protective action of GH on pituitary SS binding may study that pups immunized with GHRH-Ab are com- account for the partial preservation of the GH-releasing pletely unresponsive to exogenous GHRH immediately action of GHRH in these pups. However, regardless of this posttreatment. Ironically, in pups undergoing combined problem, it is apparent that in GHRH-Ab-treated pups the GHRH-Ab and GH treatment, whose GH release should decreased SS binding is not due to the lack of GHRH. be even worse than that of rats treated with GHRH-Ab Administration of GH in both GHRH-Ab-treated and alone, the GH response to GHRH was partially pre- control rats failed to modify baseline gene expression of served. Some morphologic features of the somatotrophs GHRH and SS. This finding is in contrast to our earlier of these pups, denoting the existence of an unexpectedly study in which we observed that a 4-fold higher dose of high cellular activity, support this finding. In fact, the GH GH, administered under the same experimental condi- immunoreactivity per unit cytoplasm area was more in- tions, decreased hypothalamic GHRH gene expression in tense and the secretory granules were smaller in the cells intact pups (34). Consequently, it cannot be ruled out that of GHRH-Ab+GH-treated pups than in somatotrophs of greater GH doses or a longer treatment period may mod- the pups treated with GHRH-Ab or GH alone, thus ify hypothalamic gene expression of GHRH and SS. It is indicating a higher concentration of GH molecules inside noteworthy that replacement therapy with GH, at the the secretory granules. The marginal location of the GH dose presently used, did not affect significantly GHRH secretory granules could explain the higher GH respon- and SS gene expression in prepubertal hypothyroid rats siveness to GHRH, because marginated granules can be (41) and that GH (75 pglrat, twice daily, s.c.) also failed rapidly released (44). Also, the abundant rough endoplas- to decrease the elevated GHRH mRNA levels in adult mic reticulum suggests an intense cytoplasmic activity, hypophysectomized rats. which, however, did not lead to cell proliferation, as In conclusion, in GHRH-deprived pups, a GH replace- shown by the absence of mitotic activity demonstrated ment therapy counteracts the disrupting effect of the with the antiproliferating cell nuclear antigen immunocy- passive immunization on growth rate, IGF-I production, tochemical reaction. and pituitary SS binding and improves the ability of the Pituitary GH-immunoreactive structures were fewer gland to respond to an acute GHRH challenge. GH re- but the PRL-immunoreactive structures more numerous placement fails to revert to normal the altered gene ex- than in the corresponding control group. This seems to pression of hypothalamic GHRH and SS. Thus, the ac- indicate 1) a particularly strong suppression of the tion of GH early postnatally appears to be mainly GHRH tone, because it is known that GHRH inhibits the directed at the pituitary, although the underlying mecha- proliferation of mammotrophs in vitro (45), and 2) a nism(~)has yet to be clarified. possible shift of the somatotroph population toward mammotrophs. (46)... REFERENCES GH replacement therapy in the NRS-treated rats sup- I. Cella SG, Locatelli V, Mennini T. Zanini A. Bcndotti C, Forloni GL. Fuma- pressed the GH response to GHRH. This is in agreement galli Ci, Arce VM, De Gennaro Coh~naV, Wehrenberg WB, Muller EE IYYU Deprivation of growth hormone-releasing hormone early in the rat's neonatal with findings obtained in adult rats (47) and ~~-d~fi~i~~~life permanently affects somatotropic function. 127:1625-1634 children (48, 49). 2. 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